The invention relates to polypeptides and peptides, particularly recombinant ones, which can be used for the diagnosis of paratuberculosis in cattle and possibly of Crohn""s disease in human beings. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against paratuberculosis.
It also relates to nucleic acids coding for said polypeptides and peptides.
Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against paratuberculosis.
By xe2x80x9crecombinant polypeptides or peptidesxe2x80x9d it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression xe2x80x9crecombinant polypeptidesxe2x80x9d such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
The term xe2x80x9crecombinantxe2x80x9d indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into an expression vector used in said host.
Nevertheless, it must be understood that the polypeptides or the peptides of the invention can be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules.
The expression xe2x80x9cbiologically purexe2x80x9d or xe2x80x9cbiological purityxe2x80x9d means on the one hand a grade of purity such that the polypeptides can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants.
Paratuberculosis (Johne""s disease) has been described as one of the most serious diseases affecting the world cattle industry. This mycobacteriosis produced by M. paratuberculosis is characterized by an ileocoecal enteritis leading successively to emaciation, dysentery, cachexy and death (Chiodini R. J. et al., 1984, xe2x80x9cRuminant paratuberculosis (Johne""s disease): the current status and future prospectsxe2x80x9d, Cronell Vet. 74:218-262). Histological examination shows oedema, infiltration and thickening of the ileal mucosa, and hypertrophy and necrosis of intestinal lymphnodes. A miliary syndrome with diffused parenchima granuloma in liver, spleen and lungs is not infrequent. The high contagiousness of this disease is due to excretion of large numbers of bacteria from the intestinal tract: contaminated pastures propagate the infection, rapidly producing live-stocks wherein infected animals represent a large part of the population. Chronical dysentery is an advanced stage of the disease, for epidemiological data suggest that the subclinical cases, with little sign of intestinal alteration correspond to the majority of infected animals and frequently to a large proportion of a live-stock population.
Diagnosis of paratuberculosis is essential, especially in the absence of clinical symptoms: it leads to identification of hidden bacterial shedders and avoids propagation of infection. Unfortunately, diagnostic indicators for early stages of the disease are missing. In fact, identification of the etiological agent (a slow grower) is a lengthy process, and histological examination of biopsy material is difficult and expensive. More interesting appear to be the immunological procedures for analysis of humoral immune reactions (Brugxc3xa8re-Picoux J., 1987, xe2x80x9cLe diagnostic de la paratuberculose chez les ruminantsxe2x80x9d, Rec. Mxc3xa9d. Vxc3xa9t. 163:539-546 Colgrave J. S. et al., 1989, xe2x80x9cParatuberculosis in cattle: a comparison of three serologic tests with results of fecal culturexe2x80x9d, Veterinary Microbiology 19:183-187). Although complement fixation and hemagglutination tests apparently lack both sensitivity and specificity, immunoenzymometric methods for evaluation of antimycobacterial antibodies seem to be more promising (Abbas B. et al., 1983, xe2x80x9cIsolation of specific peptides from Mycobacterium paratuberculosis protoplasm and their use in an enzyme linked immunosorbent assay for the detection of paratuberculosis (Johne""s disease) in cattlexe2x80x9d, Am. J. Vet. Res. 44:2229-2236; Colgrave J. S. et al., 1989, xe2x80x9cParatuberculosis in cattle: a comparison of three serologic tests with results of fecal culturexe2x80x9d Veterinary Microbiology, 19:183-187; Yokomizo Y. et al., 1983, xe2x80x9cEnzyme-linked immunosorbent assay for detection of bovine immunoglobulin Gl antibody to a protoplasmic antigen of Mycobacterium paratuberculosisxe2x80x9d Am. J. Vet. Res. 44:2205-2207; Yokomizo Y. et al., 1985, xe2x80x9cA method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine paratuberculosisxe2x80x9d Japan, J. Vet. Sci. 47:111-119).
Moreover, since slaughtering of cattle affected by tuberculosis (caused by M. bovis and/or M. tuberculosis), but not of those with paratuberculosis, is compulsory in Occidental countries, a distinction at the immunological level between the two mycobacterial diseases is essential. Moreover, M. paratuberculosis is known to be genetically close-related to M. avium (Chiodini R. J. et al., 1989, xe2x80x9cThe genetic relationship between Mycobacterium paratuberculosis and the M. avium complexxe2x80x9d Acta Leprol. 7:249-251; Hurley S. S. et al., 1988, xe2x80x9cDeoxyribonucleic acid-relatedness of Mycobacterium paratuberculosis to others members of the family Mycobacteriaceaexe2x80x9d Int. J. Syst. Bacteriol. 38:143-146), which is a possible host of the intestinal tract of ruminants.
Taking into account the cross reactivity between M. paratuberculosis and many other mycobacteria, it was a priori a difficult approach to find an antigen containing specific epitopes liable to be used as reagents for the diagnosis of paratuberculosis, said reagents having no cross reactivity with other close related mycobacteria.
In addition to the above-mentioned aspects relative to paratuberculosis in cattle, M. paratuberculosis has been found to play an etiologic role in at least some cases of Crohn""s disease in human.
The disease originally described by Crohn and coworkers was a chronical ileitis producing hyperplastic granulomata of the intestine and lymphnodes. The syndrome presently known as Crohn""s disease entails inflammatory alterations of different organs of the digestive tract (month, larynx, esophagus, stomach, ileum and colon). Segments of the motive apparatus (joints, muscles and bones) can also be involved. Isolation of mycobacteria from patients affected by the Crohn""s disease has been repeatedly related: in several instances isolates were identified as M. paratuberculosis. The induction by these isolates of a syndrome mimicking Crohn""s disease in laboratory animals and primates has been successful. In a recent review article (Chiodini R. J., 1989, xe2x80x9cCrohn""s disease and the mycobacterioses: a review and comparison of two disease entitiesxe2x80x9d, Clin. Microbiol. Rev. 2:90-117), Chiodini suggests this syndrome to be the expression of several pathological entities and concludes, that, if Crohn""s disease has a mycobacterial etiology, the most likely agent would be M. paratuberculosis. 
At this present time, larger epidemiological investigation with an ELISA based on a specific protein of M. paratuberculosis is expected to help to solve the problem of the etiology of this enteritis resembling in many respects the Johne""s disease of cattle.
The expression xe2x80x9ccattlexe2x80x9d means ruminants, such as bovines, sheeps, goats, cervidae, but also include some non ruminant animals which may also be infected by Johne""s disease such as monkeys and horses.
An aspect of the invention is to provide recombinant polypeptides which can be used as purified antigens for the detection and control of paratuberculosis.
Another aspect of the invention is to provide nucleic acids coding for the peptidic chains of biologically pure recombinant polypeptides which enable their preparation on a large scale.
Another aspect of the invention is to provide antigens which can be used in serological tests as an in vitro rapid diagnosis of paratuberculosis, as well as in skin tests for in vivo diagnosis of paratuberculosis and as an immunogenic principle in vaccines.
Another aspect of the invention is to provide a rapid in vitro diagnostic means for paratuberculosis, enabling it to discriminate between cattle suffering from tuberculosis from the ones suffering from paratuberculosis.
Another aspect of the invention is to provide a rapid in vitro diagnostic means for paratuberculosis, enabling it to discriminate between cattle suffering from paratuberculosis from the ones infected or colonized by M. avium, M. bovis or M. tuberculosis or M. phlei. 
Another aspect of the invention is to provide in vitro diagnostic means for patients suffering from Crohn""s disease.
The invention relates to an antigen complex from M. paratuberculosis, named hereafter xe2x80x9cthe antigen A36xe2x80x9d, liable to be obtained as follows:
sonication of bacterial suspensions of M. paratuberculosis to obtain a homogenate (also named sonicate),
centrifugation of the above-mentioned homogenate to obtain a supernatant (which corresponds to the cytoplasm of the bacteria),
RNAase digestion of the above-mentioned supernatant,
fractionation of the digested supernatant, for instance by gel exclusion chromatography, for instance on Sepharose 6B columns,
recovery of the antigen complex (A36) which is the excluded fraction of the fractionation.
It is to be noted that the antigen complex hereabove defined corresponds to the TMA complex (thermostable macromolecular antigens), belonging to a family of complexes present in all mycobacteria and consisting of or containing lipid, polysaccharide and protein moieties.
The proteic part of the antigen complex of the invention can be fractionated and visualized as follows:
fractionation of the proteins of the above-mentioned antigen complex by electrophoresis in a gel, for instance 10% polyacrylamide gels to obtain the protein on bands,
detection of the proteins by staining for instance with Coomassie blue.
The polypeptides of the invention contain in their polypeptidic chain:
the amino acid sequence of 101 amino acids of FIG. 8,
or a fragment of this sequence, this fragment being such that:
it is liable to be recognized by antibodies also recognizing the abovesaid sequence of 101 amino acids, but it is not recognized by antibodies raised respectively against M. bovis, M. avium, M. phlei and M. tuberculosis, 
it is liable to generate antibodies which also recognizing the abovesaid sequence of 101 amino acids but which do not recognize M. bovis, M. avium, M. phlei and M. tuberculosis, 
it reacts with the majority of sera from cattle suffering from Johne""s disease,
or the polypeptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the above-mentioned properties.
Recognition of one of the above-mentioned fragments by the above-mentioned antibodiesxe2x80x94or of the abovesaid sequence of 101 amino acids by the above-mentioned antibodiesxe2x80x94means that the above-mentioned fragment can form a complex with one of the above-said antibodies.
The formation of the complex antigen (i.e. the sequence of 101 amino acids (SEQ ID NO:5) or of the above-said fragment)xe2x80x94antibody and the detection of the existence of a formed complex can be done according to classical techniques such as the ones using a marker labeled by radioactive isotopes or by an enzyme.
Hereafter is also given in a non limitative way, a test for giving evidence of the fact that polypeptides of the invention are recognized selectively by the majority of the sera from cattle suffering from Johne""s disease (immunodominant polypeptides), for instance bovines.
This test is an immunoblotting (Western blotting) analysis, in the case where the polypeptides of the invention are obtained by recombinant techniques. This test can also be used for polypeptides of the invention obtained by a different preparation process. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polypeptides of the invention are blotted onto nitrocellulose membranes (Hybond C. (Amersham)) as described by Towbin H. et al., 1979, xe2x80x9cElectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applicationsxe2x80x9d, Proc. Natl. Acad. Sci. USA 76:4350-4354. The expression of polypeptides of the invention fused to xcex2-galactosidase in E. coli Y1089, is visualized by the binding of a polyclonal rabbit anti-A36 antiserum (or polyclonal rabbit anti-homogenate antiserum defined hereafter in the examples, or polyclonal rabbit anti-xcex2gal-p362 antiserum, defined hereafter in the examples) (1:1,000) or by using a monoclonal anti-xcex2-galactosidase antibody (Promega). The secondary antibody (anti-rabbit immunoglobulin G and anti-mouse immunoglobulin G respectively, both alkaline phosphatase conjugated) is diluted as recommended by the supplier (Promega). Colour reaction is developed by adding NBT/BCIP (Nitro Blue Tetrazolium 5-bromo 4-chloro-3-indolyl phosphate [Promega]) using conditions recommended by suppliers.
In order to identify selective recognition of polypeptides of the invention and of fusion proteins of the invention by sera of bovine suffering from Johne""s disease, nitrocellulose sheets are incubated overnight with each of these sera (1:50) (after blocking a specific protein-binding sites).
Reactive areas on the nitrocellulose sheets are revealed by incubation with peroxidase conjugated goat anti-bovine immunoglobulin G antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 h, and after repeated washings, color reaction is developed by adding xcex1-chloronaphtol (Bio-Rad Laboratories, Richmond, Calif.) in the presence of hydrogen peroxide.
The non-recognition of the antibodies raised against the above-mentioned fragments of the invention by M. bovis, M. avium, M. phlei and M. tuberculosis and by other mycobacteria can be done according to a process detailed in the examples.
As to the non-recognition of the above-mentioned fragments of the invention by antibodies raised respectively against M. bovis, M. avium, M. phlei and M. tuberculosis or other mycobacteria, it can also be done according to a process detailed in the examples.
Advantageous above-defined fragments of the invention are liable not to be recognized by antibodies raised against other mycobacteria such as M. leprae, M. intracellulare, M. scrofulaceum, M. fortuitum, M. gordonae and M. smegmatis, and are liable to generate antibodies which do not recognize M. leprae, M. intracellulare, M. scrofulaceum, M. fortuitum, gordonae and M. smegmatis. 
It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the polypeptides of the invention, particularly the free carboxyl groups which are carried by the groups Glu and Asp or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acids inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide.
The molecules which are thus modified are naturally part of the invention. The above mentioned carboxyl groups can be acylated or esterified.
Other modifications are also part of the invention. Particularly, the amine or carboxyl functions or both of terminal amino acids can be themselves involved in the bond with other amino acids. For instance, the N-terminal amino acid can be linked to the C-terminal amino acid of another peptide comprising from 1 to several amino acids.
Furthermore, any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above mentioned properties of said polypeptides.
The polypeptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
An advantageous recombinant polypeptide of the invention is constituted by the sequence represented on FIG. 8, extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (101), or by the following peptides:
Glu-Phe-Pro-Gly-Gly-Gln-Gln-His-Ser-Pro-Gln, (position 1 to 11 on FIG. 8) (SEQ ID NO:12)
Gln-Gln-Ser-Tyr-Gly-Gln-Glu-Pro-Ser-Ser-Pro-Ser-Gly-Pro-Thr-Pro-Ala (position 85 to 101 on FIG. 8) (SEQ ID NO:13).
It is to be noted that this polypeptide is derived from the expression product of a DNA derived from the nucleotide sequence coding for a polypeptide of 10 kDa being the carboxy terminal part of a 34 kDa protein of M. paratuberculosis, defined hereafter.
An advantageous recombinant polypeptide of the invention is characterized by the fact that:
it contains the amino sequence of 101 amino acids of FIG. 8 (SEQ ID NO: 5) as its C-terminal part,
it has a molecular weight of about 34 kDa, in SDS-PAGE,
it is coded by a nucleotide sequence liable to hybridize with the complementary strand of the sequence of FIG. 11 (SEQ ID NO:2),
it reacts with the majority of sera from cattle suffering from Johne""s disease,
it is advantageously liable to elicit a cellular immune response in sensitized subjects.
Subjects can be either test animals such as mice or guinea pigs or cattle or human beings.
xe2x80x9cSensitizedxe2x80x9d means that these subjects have been in contact previously with M. paratuberculosis, resulting in a priming of the cellular immune system.
Sensitization can be induced by inoculating the subjects with killed or attenuated M. paratuberculosis or it can result from a natural infection with M. paratuberculosis. 
A positive cellular immune response to the polypeptides of the invention can be detected for example in vivo by a delayedxe2x80x94type hypersensitivity reaction upon skintesting with the polypeptides of the invention or in vitro by proliferation of peripheral blood lymphocytes isolated from sensitized subjects, in response to the added polypeptides.
An advantageous recombinant polypeptide of the invention contains or is constituted by the amino acid sequence of FIG. 11 (SEQ ID NO:11).
Another advantageous recombinant polypeptide of the invention contains or is constituted by the amino acid sequence extending from amino acid at position (1) to the amino acid at position (199), of FIG. 11 (SEQ ID NO:11).
It is to be noted that this polypeptide is a 34 kDa protein which is present in the proteic part of the TMA complex of M. paratuberculosis (A36).
Hereafter is given, in a non limitative way, a process for preparing this 34 kDa protein of the invention.
The DNA sequence (306 bp) coding for p362 (SEQ ID NO:4), being the carboxyterminal end of the 34 kDa protein has been determined (SEQ ID NO:5) (see FIG. 8). It contains a unique ApaI (GGGCCC) site at position 141.
Using this information, the full gene coding for the 34 kDa protein can be isolated as follows:
An oligonucleotide coding for a stretch of at least 30 bp, situated within the region EcoRI-ApaI (1-141 bp) of the known sequence, is synthesized.
It is labeled and used as a probe to hybridize to the DNA of M. paratuberculosis (strain ATCC 19698), which has previously been cut by ApaI, separated by agarose gel electrophoresis, denatured and transferred to a nylon membrane.
This hybridization indicates a band on the nylon membrane of around 1500 bp, which contains the coding part for the rest of the 34 kDa protein. After having located this 1500 bp fragment, flanked by 2 ApaI sites, in the agarose gel, it is isolated from the gel, purified and subcloned in the ApaI site of the sequencing vector pBluescript SK+.
After sequencing of this fragment, the coding region, starting with the initiation codon ATG or GTG, is delineated. Using a restriction site near the initiation codon (5xe2x80x2 end), naturally present or created by site-directed mutagenesis, and the ApaI site at the 3xe2x80x2 end, the DNA fragment coding for the N-terminal part of the protein (about 750 bp) is excised from pBluescript SK+, and purified. It is ligated to the ApaI site of the fragment coding for the C-terminal part of p362 (142-306, FIG. 8), that for example has been prepared synthetically.
The complete gene coding for the 34 kDa protein (about 910 bp) is subcloned in an expression vector and expressed in E. coli. The recombinant 34 kDa protein is then purified.
The invention also relates to the amino acid sequences constituted by the above mentioned polypeptides and a protein or an heterologous sequence with respect to said polypeptide, said protein or heterologous sequence comprising for instance from about 1 to about 1100 amino acids. These amino acid sequences will be called fusion proteins.
In an advantageous fusion protein of the invention, the heterologous protein is xcex2-galactosidase.
The invention also relates to a nucleic acid characterized by the fact that it comprises or is constituted by:
a nucleotide chain liable to hybridize with the nucleotide chain coding for the polypeptides according to the invention, or
a nucleotide chain coding for the polypeptides according to the invention, or
the complementary sequences of the above nucleotide chains.
The invention also relates to nucleic acids comprising nucleotide sequences which hybridize with the nucleotide sequences coding for any of the above mentioned polypeptides under the following hybridization conditions:
hybridization and wash medium:
a preferred hybridization medium contains about 3xc3x97SSC [SSC=0.15 M sodium chloride, 0.015 M sodium citrate, pH 7], about 25 mM of phosphate buffer pH 7.1, and 20% deionized formamide, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinylpyrrolidone and about 0.1 mg/ml sheared denatured salmon sperm DNA,
a preferred wash medium contains about 3xc3x97SSC, about 25 mM phosphate buffer, pH 7.1 and 20% deionized formamide;
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by x-y: i.e. by the sequence extending from the extremity constituted by the nucleotide at position (x) to the extremity constituted by the nucleotide at position (y) represented on FIGS. 7A (SEQ ID NO:1), 7B (SEQ ID NO:2) or 7C (SEQ ID NO:3):
1-306 (for FIGS. 7B (SEQ ID NO:16) and 7c (SEQ ID NO:17)) or
HT=WT=65xc2x0 C.
1-307 (for FIG. 7A (SEQ ID NO:14))
1-507 (for FIGS. 7B (SEQ ID NO:2) and 7c (SEQ ID NO:3))
HT=WT=65xc2x0 C.
1-508 (for FIG. 7A (SEQ ID NO:1))
The above mentioned temperatures are to be considered as approximately xc2x15xc2x0 C.
It is to be noted that in the above defined nucleic acids, as well as in the hereafter defined nucleic acids, the nucleotide sequences which are brought into play are such that T can be replaced by U.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (307) (SEQ ID NO:14) represented in FIG. 7A,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (508) (SEQ ID NO:1) represented in FIG. 7A, wherein
X and E represent phosphodiester bonds,
Y and F represent respectively G and C,
Z and H represent respectively C and G, or
X and E represent respectively G and C,
Y and F represent respectively C and G,
Z and H represent phosphodiester bonds.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (306) (SEQ ID NO:15) represented in FIG. 7B,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (507) represented in FIG. 7B (SEQ ID NO:2).
The nucleotide sequence represented in FIG. 7B corresponds to the one represented in FIG. 7A, wherein
X and E represent phosphodiester bonds,
Y and F represent respectively G and C,
Z and H represent respectively C and G.
The invention also relates to a nucleic acid characterized by the fact that it comprises or is constituted by a nucleotide chain,
extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (306) (SEQ ID NO:16) on FIG. 7C, or
extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (507) (SEQ ID NO:3) on FIG. 7C.
The nucleotide sequence represented on FIG. 7C corresponds to the one represented on FIG. 7A, wherein
X and E represent respectively G and C,
Y and F represent respectively C and G,
Z and H represent phosphodiester bonds.
The invention also relates to a nucleic acid which comprises or is constituted by:
a nucleotide sequence liable to hybridize with the complementary strand of the nucleotide sequence of FIG. 11 (SEQ ID NO:10), or with the complementary strand of the nucleotide sequence extending from nucleotide at position (742) to nucleotide at position (1338) (SEQ ID NO:17) of FIG. 11,
the nucleotide sequence of FIG. 11 or the nucleotide sequence extending from nucleotide at position (742) to nucleotide at position (1338) (SEQ ID NO:17) of FIG. 11,
the complementary sequences of the nucleotide sequences above-defined.
From the nucleic acids of the invention, probes (i.e. cloned or synthetic oligonucleotides) can be inferred.
These probes can be from 15 to the maximum number of nucleotides of the selected nucleic acids. The oligonucleotides can also be used either as amplification primers in the PCR technique (PCR, Mullis and Faloona, Methods in Enzymology, vol. 155, p. 335, 1987) to generate specific enzymatically amplified fragments and/or as probes to detect fragments amplified between bracketing oligonucleotide primers.
The specificity of a PCR-assisted hybridization assay can be controlled at different levels.
The amplification process or the detection process or both can be specific. The latter case giving the higher specificity is preferred.
The invention also relates to any recombinant nucleic acid containing at least one of the nucleic acids of the invention combined to or inserted in a heterologous nucleic acid.
The invention relates more particularly to recombinant nucleic acid such as defined, in which the nucleotide sequence of the invention is preceded by a promoter (particularly an inducible promoter) under the control of which the transcription of said sequence is liable to be processed and possibly followed by a sequence coding for transcription termination signals.
The invention also relates to the recombinant nucleic acids in which the nucleic acid sequences coding for the polypeptide of the invention and possibly the signal peptide, are recombined with control elements which are heterologous with respect to the ones to which they are normally associated with in the mycobacterial genome and, more particularly, the regulation elements adapted to control their expression in the cellular host which has been chosen for their production.
The invention also relates to recombinant vectors, particularly for cloning and/or expression, comprising a vector sequence, notably of the type plasmid, cosmid or phage or virus DNA, and a recombinant nucleic acid of the invention, inserted in one of the non essential sites for its replication.
According to an advantageous embodiment of the invention, the recombinant vector contains necessary elements to promote the expression in a cellular host of polypeptides coded by nucleic acids according to the invention inserted in said vector and notably a promoter recognized by the RNA polymerase of the cellular host, particularly an inducible promoter and possibly a sequence coding for transcription termination signals and possibly a signal sequence and/or an anchoring sequence.
According to another additional embodiment of the invention, the recombinant vector contains the elements enabling the expression by E. coli of a fusion protein consisting of the polypeptide of xcex2-galactosidase or part thereof linked to a polypeptide coded by a nucleic acid according to the invention.
The invention also relates to a cellular host, chosen from among bacteria such as E. coli or chosen from among eukaryotic organism, such as CHO cells or insect cells, which is transformed by a recombinant vector according to the invention, and containing the regulation elements enabling the expression of the nucleotide sequence coding for the polypeptide according to the invention in this host.
The invention relates to an expression product of a nucleic acid expressed by a transformed cellular host according to the invention.
The invention also relates to a process for preparing a recombinant polypeptide according to the invention comprising the following steps:
the culture in an appropriate medium of a cellular host which has previously been transformed by an appropriate vector containing a nucleic acid according to the invention,
the recovery of the polypeptide produced by the above said transformed cellular host from the above said culture medium, or from the cellular host, and
possibly the purification of the polypeptide produced, eventually by means of immobilized metal ion affinity chromatography (IMAC).
The polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled xe2x80x9cMethode der organischen chemiexe2x80x9d (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase according to the method described by Atherton and Shepard in their book titled xe2x80x9cSolid phase peptide synthesisxe2x80x9d (Ed. IRL Press, Oxford, NY, Tokyo, 1989).
The invention also relates to a process for preparing the nucleic acids according to the invention.
A suitable method for chemically preparing the single-stranded nucleic acids (containing at most 100 nucleotides of the invention) comprises the following steps:
DNA synthesis using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986.
In the case of single-stranded DNA, the material which is obtained at the end of the DNA synthesis can be used as such.
A suitable method for chemically preparing the double-stranded nucleic acids (containing at most 100 bp of the invention) comprises the following steps:
DNA synthesis of one sense oligonucleotide using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986, and DNA synthesis of one anti-sense oligonucleotide using said above-mentioned automatic xcex2-cyanoethyl phosphoramidite method,
combining the sense and anti-sense oligonucleotides by hybridization in order to form a DNA duplex,
cloning the DNA duplex obtained into a suitable plasmid vector and recovery of the DNA according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
A method for the chemical preparation of nucleic acids of length greater than 100 nucleotidesxe2x80x94or bp, in the case of double-stranded nucleic acidsxe2x80x94comprises the following steps:
assembling of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the succession of amino acids in the natural peptide, according to the principle described in Proc. Nat. Acad. Sci. USA 80; 7461-7465, 1983,
cloning the DNA thereby obtained into a suitable plasmid vetor and recovery of the desired nucleic acid according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
The invention also relates to antibodies themselves formed against the polypeptides according to the invention, and characterized by the fact that they recognize neither M. bovis, nor M. avium, nor M. phlei, nor M. tuberculosis. 
It goes without saying that this production is not limited to polyclonal antibodies.
It also relates to any monoclonal antibody produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the purified polypeptide of the invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by its ability to produce the monoclonal antibodies recognizing the polypeptide which has been initially used for the immunization of the animals.
The invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type.
The polypeptide which is advantageously used to produce antibodies, particularly monoclonal antibodies, is the one or part of the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (101) (SEQ ID NO:5) represented on FIG. 8.
Variations of this peptide are also possible depending on its intended use. For example, if the peptide is to be used to raise antisera, the peptide may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labeled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. This peptide possesses therefore the primary sequence of the peptide above-mentioned but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptide.
The invention also relates to a process for detecting in vitro antibodies related to paratuberculosis in a biological sample of an animal liable to contain them, this process comprising
contacting the biological sample with a polypeptide or a peptide according to the invention, or the expression product of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by an animal serum, and particularly by bovine serum.
The detection can be carried out according to any classical process.
By way of example a preferred method brings into play an immunoenzymatic process according to ELISA technique or immunofluorescent or radioimmunlogical (RIA) or the equivalent ones.
Thus the invention also relates to any polypeptide according to the invention labeled by an appropriate label of the enzymatic, fluorescent, radioactive . . . type.
Such a method for detecting in vitro antibodies related to paratuberculosis comprises for instance the following steps:
deposit of determined amounts of a polypeptidic composition according to the invention in the wells of a titration microplate,
introduction into said wells of increasing dilutions of the serum to be diagnosed,
incubation of the microplate,
repeated rinsing of the microplate,
introduction into the wells of the microplate of labeled antibodies against the blood immunoglobulins,
the labeling of these antibodies being based on the activity of an enzyme which is selected from among the ones which are able to hydrolyze a substrate by modifying the absorption of the radiation of this latter at least at a given wave length,
detection by comparing with a control standard of the amount of hydrolyzed substrate.
The invention also relates to a process for detecting and identifying in vitro antigens of M. paratuberculosis in an animal biological sample liable to contain them, this process comprising:
contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. paratuberculosis which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by serum or faeces, milk or urine, particularly of bovine origin.
Appropriate antibodies are advantageously monoclonal antibodies directed against the above-mentioned peptide.
The invention also relates to an additional method for the in vitro diagnosis of paratuberculosis in an animal liable to be infected by Mycobacterium paratuberculosis comprising:
contacting a biological sample taken from an animal with a polypeptide or a peptide of the invention, or the expression product of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide or peptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which has possibly been formed.
To carry out the in vitro diagnostic method for paratuberculosis in an animal liable to be infected by Mycobacterium paratuberculosis, the following necessary or kit can be used, said necessary or kit comprising:
a polypeptide or a peptide according to the invention, or the expression product of the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognized by a labeled reagent, more particularly in the case where the above mentioned polypeptide or peptide is not labeled.
The invention also relates to an additional method for the in vitro diagnosis of paratuberculosis in an animal liable to be infected by M. paratuberculosis, comprising the following steps:
contacting a biological sample of said animal with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. paratuberculosis which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
To carry out the in vitro diagnostic method for paratuberculosis in an animal liable to be infected by Mycobacterium paratuberculosis, the following necessary or kit can be used, said necessary or kit comprising:
an antibody of the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complexes which have been produced by the immunological reaction, said reagent possibly having a label or being liable to be recognized by a labeled reagent, more particularly in the case where the above-mentioned antibody is not labeled.
An advantageous kit for the in vitro diagnosis of paratuberculosis comprises:
at least a suitable solid phase system, e.g. a microtiter-plate for deposition thereon of the biological sample to be diagnosed in vitro,
a preparation containing one of the monoclonal antibodies of the invention,
a specific detection system for said monoclonal antibody,
appropriate buffer solutions for carrying out the immunological reaction between a test sample and said monoclonal antibody on the one hand, and the bonded monoclonal antibodies and the detection system on the other hand.
The invention also relates to a kit, as described above, also containing a preparation of one of the polypeptides or peptides of the invention, said antigen of the invention being either a standard (for quantitative determination of the antigen of M. paratuberculosis which is sought) or a competitor, with respect to the antigen which is sought, for the kit to be used in a competition dosage process.
The invention also relates to a method for the in vitro diagnosis of Crohn""s disease in a patient liable to be infected by Mycobacterium paratuberculosis comprising the following steps:
contacting the biological sample with an appropriate antibody according to the invention, under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. paratuberculosis which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
The invention also relates to a method for the in vitro diagnosis of Crohn""s disease in a patient liable to be infected by M. paratuberculosis, comprising the following steps:
contacting a biological sample taken from a patient with a polypeptide or peptide according to the invention, or the expression product of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which has been possibly formed.
The invention also relates to a necessary or kit for an in vitro diagnosis method of Crohn""s disease in a patient liable to be infected by Mycobacterium paratuberculosis, said necessary or kit comprising:
an antibody according to the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complexes which have been produced by the immunological reaction, said reagents possibly having a label or being liable to be recognized by a labeled reagent, more particularly in the case where the above-mentioned antibody is not labeled.
The invention also relates to a necessary or kit for an in vitro diagnosis method of Crohn""s disease in a patient liable to be infected by Mycobacterium paratuberculosis said necessary or kit comprising:
a polypeptide or a peptide according to the invention, or the expression product of the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognized by a labeled reagent, more particularly in the case where the above mentioned polypeptide is not labeled.
The invention also relates to an immunogenic composition comprising a polypeptide or a peptide according to the invention, or the expression product of the invention, in association with a pharmaceutically acceptable vehicle.
The invention also relates, to a vaccine composition comprising among other immunogenic principles anyone of the polypeptides or peptides of the invention or the expression product of the invention, possibly coupled to a natural protein or to a synthetic polypeptide having a sufficient molecular weight so that the conjugate is able to induce in vivo the production of antibodies neutralizing Mycobacterium paratuberculosis, or induce in vivo a protective cellular immune response by activating M. paratuberculosis antigen-responsive T cells.
The invention also relates to a necessary or kit for the diagnosis of prior exposure of an animal to M. paratuberculosis, said necessary or kit containing a preparation of at least one of the polypeptides or peptides of the invention, or the expression product of the invention, with said preparation being able to induce in vivo after being intradermally injected to an animal a delayed type hypersensitivity reaction, at the site of injection, in case the animal has had prior exposure to M. paratuberculosis. 
Other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention.